A STABLE COMPOSITION

Abstract: Human islet glutamic acid decarboxylase65 (GAD65) can be stabilized by adding a reducing agent, a detergent, an alcohol or a divalent metal ion.

Description: A STABLE COMPOSITION INTRODUCTION The present invention relates to a stabilization of purified recombinant human islet glutamic acid decarboxylase 65 (GAD 65 , EC 4J J J5) and to a process for preparing stable enzymatically and antigenic active GADg5- BACKGROUND OF THIS INVENTION The disease Insulin-dependent diabetes mellitus (IDDM) results from an autoimmune destruction of the insulin producing β cells of the islets of Langerhans. Epidemiological studies suggest that the incidence of the disease is increasing, at least in the USA and Europe. The disease becomes clinical manifest when about 80 % of the β-cells have been destroyed. The progressive loss of the β cell mass spans over several years raising the possibility of early treatment of the disease by saving a major part of the β cell mass. It would be desirable, therefore, to determine whether individuals were in the prediabetic phase or not. Circulating autoantibodies towards β-cell antigens have been detected in a number of newly onset diabetics (Bottazzo et aL, Lancet 1 (1980), 668 - 672; Palmer et aL, Science 222 (1983), 1137 - 1139; and Baskkeskov et aL; J.Clin.ln- yest. 79 (1987), 926 - 934). The most reliable indicator of ongoing β-cell destruction is circulating antibodies towards the 64K antigen. These antibodies have been detected in 70 - 80 % of newly diagnosed patients and up to 8 years before the clinical manifestation of the disease (Baskkeskov et aL; J.CIin.lnvest. 79 (1987), 926 - 934; and Atkinson et aL, Lancet 335 (1990), 1357 - 1360). The 64K islet cell antigen was recently identified as the enzyme glutamic acid decarboxylase, EC 4.1 J .15 (Baakkeskov et aL, Nature 347, 151 - 156) which converts L-glutamic acid into the neurotransmitter gamma-aminobutyric acid. With the recent cloning (Karlsen et aL, Proc.Natl. Acad.Sci.USA 88 (1991), 8337 - 8341), it became possible to generate large scale antigen specific assays to measure autoantibodies towards glutamic acid decarboxylase (GAD) and to test the pathogenic role of GAD in IDDM, e.g. by determination of T-cell reactivity to GAD, induction of diabetes by immunization of rodents by GAD and prevention of spontaneous diabetes in NOD mice and BB rats by induction of tolerance to GAD. A number of patent applications have been filed on using this protein for multiple purposes (Atkinson et aL, PTC patent application having publication No. WO 90/07117; Baekkeskov et aL, PTC patent application having publication No. WO 92/04632; Lernmark et aL, PTC patent application having publication No. WO 92/20811 ; Tobin et aL, European patent application having publication No. 519,469 A1 ; and Endl et aL, European patent application having publication No. 499J76 A2) based on recombinant expressed GAD in various expression systems. However, the cloning and subsequent production of large quantities of recombinant GAD is of little interest if the biological characteristics of the molecule (enzymatic activity, antigenic properties) are missing. Natural human islet GADg 5 is a complex molecule, containing 15 cysteine residues and several palmitoylated sites. In solution, GADg 5 aggregates rapidly to form both covalent and non- covalently bound multimers having reduced enzymatic activity and reduced ability to react with antibodies. These characteristics make the isolation of recombinant GAD β5 difficult. The material is often purified at high concentration (for example, on an immuneaffinity column) under which conditions its inherent tendency to irreversibly aggregate leads to the formation of unusable precipitates. The known, purified or highly purified compositions of recombinant GADg are extremely unstable in aqueous solutions resulting in loss of enzymatic activity and loss of antigenic activity. One object of this invention is to prepare stable, purified or highly purified compositions of recombinant GAD 65 SUMMARY OF THIS INVENTION The invention described herein concerns a novel stable and biologically active recombinant human islet GADg 5 composition. Purified recombinant GADg 5 is unstable in aqueous buffers and specific measures have to be taken to purify and stabilize recombinant human GADg 5 . We have, surprisingly, found that GAD 65 can be stabilized, resulting in preservation of the biological activity, by purifying GAD 65 under reducing conditions in the presence of detergents and that purified GADg can be kept biologically active under reducing conditions, preferably in the presence of a detergent, an alcohol or a divalent metal ion. Consequently, this invention relates to a glutamic acid decarboxylasegg composition containing a reducing agent and/or a detergent and/or an alcohol and/or a divalent metal ion. DETAILED DESCRIPTION OF THIS INVENTION The present invention deals with stabilization of GADg5 using specific immunological and biological activity as criteria for maintenance of stability. The stabilized GADg 5 may be obtained by adding certain components during extraction and down stream processing. Surprisingly, we have found that GADg 5 - also as a highly purified composition - can be stabilized in the presence of reducing agents and, if preferred, in the presence of a detergent, an alcohol, or a divalent metal ion or mixtures thereof. GAD is a firmly membrane anchored protein (Christgau et aL; J.Cell.Biol. 118(2) (1992), 309 - 320) and the presence of detergents under extraction and prepuri- fication may be of importance for obtaining high yields. Normally, proteins are destabilized under denaturing and reducing conditions. In contrast, GADg 5 not only keeps its specific biological activity under these abnormal conditions. Surprisingly, an increased specific activity is observed. It is a further surprising aspect of this invention that GADg 5 is stable under reducing conditions in the presence of ionic detergents like SDS. Preferably, the compositions of this invention contain between 0.01 mg/ml and 10 mg/ml, more preferred between 0J mg/ml and 3 mg/ml, and most preferably between 1 and 3 mg/ml, of GADg 5 . Preferably, the compositions of this invention contain GAD in a very pure form, preferably GAD having a purity of more than 95 % (weight/weight) relative to the total amount of protein. Earlier descriptions of GAD purification will not lead to a useful product, for example, if the source is recombinant expressed GADg 5 . The preferred reducing agent to be added to the compositions of this invention is dithiothreitol (DTT), 2-mercaptoethanol, cysteine or cystamine, for example, at concentrations from 1 to 100 mM, preferably at concentrations below 20 mM. The detergent to be added to the compositions of this invention may be non-ionic or ionic. Preferred non-ionic detergents are triton X-114 (TX-114), triton X-100 (TX- 100) or n-octyl glucoside, for example, in a concentration in the range from 0.01 to 10 % (volume/volume), preferably at a concentration in the range from 0.5 to 5 % (volume/volume). The preferred ionic detergent to be added is sodiumdodecylsulphate (SDS), for example, in a concentration in the range from 0.001 to 1 % (weight/volume), preferably in a concentration in the range from 0.01 to 0J % (weight/ volume). The preferred alcohol to be added to the compositions of this invention is glycerol or a polyethylene glycol, for example PEG 600. The alcohol may be present in a concentration in the range from 20 to 70 % (volume/volume), preferably in a concentration less than 50 % (volume/volume). The preferred divalent metal ion to be added to the compositions of this invention is Zn + + or Ni + + , for example, at concentrations from 0J to 1 mM. This invention also relates to a process for isolating or purifying GAD 65 obtained from a crude material containing GADg 5 , for example, in the range from 0J to 20 % (weight/ weight). Apart from the addition of a reducing agent, the isolation or purification of GADgg is performed in a manner known p_er se including the presence of the co-factor, PLP, for example, at 0.2 to 1.0 mM concentration. Examples of generel isolation or purification processes are extraction, phase separation, precipitation, filtration, ion exchange chromatography, gel permeation chromatography, affinity chromatography and hydrophobic interaction chromatography, which can be combined into different down stream processes. The use of such generel processes in combination to GADg 5 is known to the skilled art worker. It is preferred that the reducing agent is present during the whole isolation or purification procedure. If the reducing agent is not present during the whole isolation or purification process but is present only during the major part of the isolation or purification process, a fairly stable composition may be obtained. An embodiment of this invention is purification of GAD 65 by affinity chromatography using elution at a pH value of about 9.5 or by anion exchange chromatography in the presence of a reducing agent and/or detergents. One way of preparing the compositions of this invention is to add, to a glutamic acid decarboxylase 6 5 composition, such an amount of a reducing agent and/or a detergent and/or an alcohol and/or a divalent metal ion as is stated herein to give the desired effect. The preparation of such compositions is known to any skilled art worker. Being a major autoantigen in type 1 diabetes mellitus, the composition of this invention is of vital importance for the screening of specific autoantibodies in human serum and thereby for prediction of the disease. A major advantage of this invention is that it provides the means to produce large quantities of highly purified stable enzymatically and antigenic active GADg5 which easily can be formulated and hereafter used in screening assays, tolerance experiments, T-cell experiments, immune invention, etc. The novel GAD 65 compositions of this invention are used similar with the use of known GAD compositions, bearing the improved stability in mind. The abbreviations used herein are as follows: BHK is baby hamster kidney cells, GAD is glutamic acid decarboxylase, DTT is dithiotreitol, PLP is pyridoxal-5'- phosphate, SDS is sodium dodecyl sulphate, IDDM is insulin dependent diabetes mellitus, NOD is non-obese diabetic, BB is Bio Breeding, EGTA is ethylene glycol- bis-(β-aminoethyl ether), AET is 2-aminoethylisothiouronium bromide hydrobromide, EDTA is ethylenediaminetetraacetic acid, PEG is polyethylene glycol, IgG is immunoglobilin, ELISA is enzyme-linked immunosorbent assay, PAGE is polyacrylamide gelelectrophoresis, and s.d. is standard deviation. The following examples are offered by way of illustration, not by limitation. Example 1 : Stabilization of GAD 5 expressed in BHK cells 100 ml BHK cell carrier pellets were homogenized with a glass homogenizer in 15 ml of 10 mM Hepes, 1 mM EGTA, 1 mM MgCI 2. 0.2 mM PLP, 1 mM AET, and 100 mg/1000 ml aprotinin (pH 7.4). The extract was centrifuged at 25000 rpm (SW25) at 4°C for 30 minutes. To aliquots of 200 μl of extract was added 200 μl of 2 % (volume/volume) TX-114, 2 % (volume/volume) SDS, 2 % (volume/volume) n-octyl glucoside, 2 mM NiCI 2 , 2 mM ZnCI 2 . ethanol, PEG 600, glycerol or H 2 0 (control). After one hour, the pH value was raised to 9.5 by addition of 25 μl of 1 M NaHCO (pH 9.5) and the vials were left for 1 hour. The experiments were carried out at 4°C. To determine the enzymatic activity, 25 μl aliquots from each sample were mixed with 225 μl of 50 mM potassium phosphate, 1 mM EDTA, 1 % TX-114, 0.2 mM PLP, and 1 mM AET (pH 7.2). Aliquots were taken immediately before raising the pH value to 9.5 and after 1 hour's exposure to a pH value of 9.5. Samples for enzyme assay were stored at -20°C prior to analysis. The enzymatic activity of the control at a pH value of 7.4 was set to 100 %. All other activities were calculated relative to this sample. The experiment was repeated under reducing conditions, 10 mM DTT (see Table 1 , below). 10 rat brains were homogenized with a polytrone in 100 ml of 10 mM Hepes, 1 mM EGTA, 1 mM MgCI 2 , 0.2 mM PLP, 1 mM AET, and 100 mg/1000 ml aprotinin (pH 7.4) and filtered on a 0.45 μ Nalgene filter. This extract was used as a reference material and analyzed as described above except that 180 μl of extract was mixed with 20 μl of 10 % solutions of TX-114, SDS, n-octyl glucoside (NOG), 10 mM solutions of NiCI 2 and ZnCI 2 and that 200 μl of ethanol, PEG 600 and glycerol were mixed with 200 μl of extract (see Table 2, below). Table 1 : BHK cell GADg 5 stability in presence of various additives. Additives - DTT, - DTT, + DTT, + DTT, pH 7.4, pH 9.5, pH 7.4, pH 9.5, in % in % in % in % 1 % TX-114 110 94 150 130 1 % SDS 0 0 0 0 1 % NOG 100 92 140 120 1 mM NiCI 2 96 89 140 100 1 mM ZnCI 2 69 88 120 160 50 % ethanol 30 0 110 100 50 % PEG 600 93 0 130 110 50 % glycerol 69 110 140 130 H 2 0 100 90 140 120 Table 2: Rat brain GADgg stability in presence of various additives. Additives - DTT, - DTT, + DTT, + DTT, pH 7.4, pH 9.5, pH 7.4, pH 9.5, in % in % in % in % 1 % TX-114 100 67 143 132 0.1 % SDS 112 78 152 163 1 % SDS 0 0 0 0 1 % NOG 105 71 138 138 1 mM NiCI 2 88 79 129 139 1 mM ZnCI 2 83 64 136 145 50 % ethanol 76 0 81 12 50 % PEG 600 109 0 129 88 50 % glycerol 112 114 140 174 H 2 0 100 64 138 148 All enzymatic activities were calculated relative to the activity of GADg at a pH value of 7.4 (control). Data are average of two experiments. Example 2: Elution of GADg5 from GAD1-Sepharose-4B matrix 5 150 μl of GAD1 -Sepharose-4B (coupled according to manufactures manual, Pharmacia, Sweden; Dr. A. Tobin, GAD1 :ATCC HB184) were washed twice in 10 mM Hepes, 10 mM cysteine, 10 mM benzamidine, 1 mM EDTA, 1 mM PLP, 1 mM AET, 1 mM glutamate, and 2 % (volume/volume) TX-114 (pH 7.4) and mixed with 1.0 ml of extract from BHK cell culture. After 1 hour's gentle rotation at 4°C, the 0 supernatant was discarded and the matrix washed 3 times in 20 mM Hepes, and 5 mM DTT (pH 7.4). GADg 5 was eluted with 500 μl of the respective elution buffers (see Table 3, below). 25 μl of the eluate was diluted into 225 μl of 50 mM potassium phosphate, 1 mM EDTA, 1 % TX-114, 0.2 mM PLP, and 1 mM AET (pH 7.2) for enzyme assay. 5 Table 3: Elution of GADg 5 from GAD-1-Sepharose 4B. Buffer no. mU eluted 1 1.22 2 2.62 0 3 5.50 4 4.41 5 2.50 6 2.73 7 2.37 5 In total, 4J3 units were applied the matrix. The buffers in Table 3 represent: 50 mM glycine buffer at pH 9.5 (1); glycine buffer with: 10 mM DTT (2); 10 mM DDT, 1 mM PLP (3); 10 mM DTT, 1 mM PLP, 1 % n-octyl glucoside (4); 10 mM DTT, 1 mM PLP, 1 % n-octyl glucoside, 1 mM NiCI 2 (5); 10 mM DTT, 1 mM PLP, 50 % glycerol (6); 10 mM DTT, 1 mM PLP, 1 mM NiCI 2 , 50 % glycerol (7). Example 3: Purification of BHK cell expressed GAD g: Yields and specific activities 100 ml of BHK-carrier pellet were mixed with 100 ml of 40 mM Hepes, 20 mM cysteine, 20 mM benzamidine, 2 mM EDTA, 2 mM PLP, 2 mM AET, 2 mM glutamate, and 2 % (volume/volume) TX-114 (pH 7.4) for 1 hour at 4°C. The extract was centrifuged for 10 minutes at 3300 x g at 4°C. The supernatant was prefiltered on (AP2504200, Millipore) followed by filtration on a 1.2 μ filter (RAWP 04700, Millipore). The temperature was raised to 37°C in a water bath and the filtrate was centrifuged at 3300 x g for 5 minutes at 37°C. The aqueous phase was extracted by adding TX-114 to 1 % (volume/volume), raising the temperature to 37°C and centrifugation as described above. The combined detergent was cooled to 4°C, diluted 4-fold with 20 mM Hepes, 10 mM cysteine, 10 mM benzamidine, 1 mM EDTA, 1 mM PLP, 1 mM AET, and 1 mM glutamate (pH 7.4) and applied (0J5 ml/min) to a 1.6 x 1.5 cm GAD1-Sepharose-4B affinity column (5 mg GAD1/ml gel) with a 1.5 x 1.0 cm Sephadex G25 (Pharmacia, Sweden) precolumn. The column system was preequilibrated in 20 mM Hepes, 10 mM cysteine, 10 mM benzamidine, 1 mM EDTA, 1 mM PLP, 1 mM AET, 1 mM glutamate, and 1 % (volume/volume) TX-114 (pH 7.4) and then washed in 20 mM Hepes, 10 mM DTT, and 1 mM PLP (pH 7.4). GAD 65 was eluted with 50 mM glycine, and 10 mM DTT, (pH 9.5) into 200 μl of 0.5 M Hepes (pH 7.0). Fractions of 0.8 ml GAD 65 positive fractions were pooled and stored at -20°C in aliquots of 500 μl. Yields and specific enzymatic activities are shown in Table 4, below. Table 4: Purification of GADg 5 from BHK cell culture - yields. Step volume μg/ml μg mU/ml mU Yield mU/mg Extract 110 6.3 693 2.6 286 100 0.41 Filtrate 105 7.4 777 3.0 315 110 0.41 AQ 85 0.38 32 0J2 10 3.5 0.31 DP 100 5.4 540 2.3 230 80 0.43 Pool 5.5 67 369 25.4 140 49 0.38 Data are based on extraction and purification of one batch of 100 ml BHK-carrier pellet. The amounts of GAD 65 were calculated from ELISA using 2U = 1 μg. The yield of GAD 65 was calculated relative to the bioactivity in the initial extract. 1 mU is defined as 1 nmol substrate converted per minute at 37°C. AQ is aqueous phase and DP is detergent phase. Example 4: Purification of GAD expressed in SF9 insect cells Cells were extracted (cells from 200 ml culture in 20 ml extraction buffer) by Dounce homogenization at 4°C in 40 mM HEPES, 5 mM DTT, 5 mM EDTA, and 50 μM pyridoxal phosphate, (pH 7J), containing 20 % (volume/volume) precondensed Triton X-114, and the protease inhibitors used in immunoprecipita- tion. The extract was centrifuged at 100,000 x g for 30 minutes at 4°C. The pellet was reextracted and the extract centrifuged as above. The pooled supernatants were heated to 30°C for one to 2 minutes to induce phase separation of the Triton X-114 and centrifuged at 4000 x g at 30°C for 10 minutes. The detergent- rich lower phase was collected. Ten per cent (volume/volume) precondensed Triton X-114 was added to the upper aqueous phase that was then cooled to 4°C and then phase separated as above. The two detergent-rich phases were pooled, diluted twice in extraction buffer and stored at -80°C. GAD 65 was isolated from the detergent extract by ion-exchange chromatography at 4°C on a 2 cm 2 x 6 cm column of DEAE Superose High Flow. Yields and specific enzymatic activities are shown in Table 5, below. Table 5: Purification of SF9 insect cell expressed GAD 65 by ion exchange chromatography. Step Vol¬ μg/ml μg mU/ml mU yield, mU/mg ume in % Extract 90 393.3 35400 175.5 15800 100 0.45 AQ 65 57 3705 16.9 1100 6.7 0.3 DP 90 240 21600 74.4 6700 42.4 0.31 Pool 46 342.2 15744 113 5200 32.9 0.33 Data from 600 ml culture of SF9 cells harvested 4 days Post Infection with recombinant virus. Protein assayed by fluorometric method as 350 μg/ml. The yield of GAD 65 was calculated relative to the bioactivity in the initial extract. 1 mU is defined as 1 nmol substrate converted per minute at 37°C. AQ is aqueous phase and DP is detergent phase. Example 5: Measurement of enzymatic activity. The enzymatic activity of GAD was assayed according to Wu J-Y. et aL; Methods in Enzvmoloqy 113 (1985), 3 - 10. In brief, the incubation tube (Sarstedt 1.5 ml) contained 4 μl of L-(1- 14 C) glutamic acid (Amersham, U.K.) in 50 mM potassium phosphate, 1 mM EDTA, 1 % TX-114, 0.2 mM PLP, 1.0 mM AET (pH 7.2) and 21 μl of 5 mM L-glutamate (105 nmol). The reaction was initiated by adding 125 μl of sample or enzyme standard. 50 μl of Hyamine base (Packard 6006005) were pipetted into a filter disk (Whatman) placed in the lid. Immediately thereafter, the tubes were vortexed, closed with the filter and incubated first for two hours at 37°C and then for 60 minutes at 4°C. The filters were transferred to 2 ml of Ultima Gold scintillation liquid (Packard) and counted in a Liquid scintillation β-counter (Tri-carb 4530, Packard). One enzyme unit is defined as 1 μmol of product formed per minute at 37°C. Example 6: EUSA for human GAD^ The assay was carried out at room temperature. 96-plates (Nunc Immuno-plates, Denmark) were coated overnight with 100 μl/well of 5 μg/ml of purified GAD1 in 10 mM sodium phosphate, and 0J M NaCI (pH 6.5). The plates were then blocked in 10 mM sodium phosphate, 0J M NaCI, 0.25 % Tween 20, and 1 % (weight/volume) bovine serum albumin (pH 6.5). Reference and unknown samples were diluted in BSA buffer containing 2 % (volume/volume) Triton X-100 and 100 μl aliquots added to the wells, incubated for one hour and washed three times. Biotinylated GAD6 (12.5 ng/ml in blocking buffer; Dr. D.I. Gottlieb and the Development Studies Hybridoma Bank, N01-HD-6-2915) was added to the plates, incubated for 1 hour, and the plates were washed three times before peroxidase labelled streptavidin (Amersham, U.K.) was added to the wells. Finally, after one hour of incubation, the wells were washed four times and peroxidase substrate 1 ,2-phenyldiamine dihydrochloride (DAKO, Denmark) dissolved in 72.9 mM sodium citrate, and 4.2 mM sodium borate (pH 5.0), was added. The incubation was stopped after 10 minutes by the addition of 2N sulphuric acid and the optical density was measured at 490 and 650 nm. The concentration of GAD 65 was determined by ELISA analysis using recombinant human islet GADg 5 isolated from SF9 cells. 1 μg of protein equals 2 units as calculated from amino acid analysis of the said material. Example 7: 1 D-SDS Electrophoresis and Western Blotting One-dimensional polyacrylamide electrophoresis was carried out on a Mini- Protean ® II Dual Slab Cell (Bio-Rad) using 10 % SDS-PAG Plate 10 gels (Daiichi Pure Chemicals Co., LTD, Japan) according to Laemmli (Nature 227 (1970), 680 - 685). Samples were prepared by adding 1 part of sample buffer (0.1 M Tris, 10 % SDS, and 50 % glycerol (pH 8.0)) to 4 parts of sample. Aliquots from 10 to 30 μl hereof were applied directly (non-reducing conditions) or boiled for 1 minute in the presence of 0.11 M DTT (reducing conditions). Staining of gels were performed with Coomassie Brilliant Blue R-250 in 40 % (volume/volume) methanol, and 10 % (volume/volume) acetic acid. 1 D-SDS PAGE of BHK cell extract, aqueous phase, detergent phase and purified GADg 5 under reducing and non-reducing conditions is shown in Figure 1. For Western blot analysis, proteins were transferred to PVDF membranes (Immobilon, Millipore) by electroblotting. The membranes were incubated in 50 mM Tris, 0.15 M NaCI, 0.1 % (volume/volume) Tween 20, and 3 % (weight/volume) milk powder (pH 7.4) for 30 minutes, further incubated for 1 hour in rabbit antiserum (R1267, Petersen et aL; Diabetes 42 (1993), 484 -495) in the incubation buffer (dilution 1 :200), washed three times in 50 mM Tris, and 0.15 M NaCI (pH 7.4) and finally incubated with alkaline phosphatase labelled goat anti- rabbit IgG (Zymed) for 1.5 hours in the incubation buffer (dilution 1 :1000). After repeated washing, the membrane was then visualized with nitroblue tetrazolium (Sigma) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma) in 0.1 M Tris, 0.1 M NaCI, and 5 mM MgCI 2 (pH 9.5). Western blot analysis of human islet extract and BHK cell extract under non-reducing conditions is shown in Figure 2. Example 8: Immune analysis of recombinant human islet GAD^ 5 To prepare a radioactively labelled GAD 65 tracer, the cDNA coding for human islet GAD β5 was cloned into the KH1117 transcription vector between a T7 RNA polymerase promotor and a poly A tail and two T7 transcription stop codons in tandem. The cDNA was transcribed in vitro according to the manufactures instructions (Promega, Madison) and translated in vitro in the presence of ^ 5 S- 10 methionine (Amersham, U.K.) using a rabbit reticulocyte lysate (Promega) and the final product desalted using a NAP5 column (Pharmacia, Sweden). To test for the presence of GAD autoantibodies in sera, 35 S-methionine GAD 65 (approx. 35000 cpm) was incubated overnight at 4°C with serum (final dilution 1 :25) in a total volume of 50 μl of 50 mM Tris, 150 mM NaCI, and 0J % (volume/volume) Tween- 15 20 (pH 7.4). Immune complexes were precipitated by incubation with 7.5 mg of Protein-A Sepharose (Pharmacia, Sweden) in 50 μl of 30 mM Tris, 150 mM NaCI, and 0.1% (volume/volume) Tween 20 (pH 7.4) for 2 hours at 4°C. Finally, the reaction volume was transferred to a 96-well Multiscreen filtration system (Millipore) followed by 10 times wash of the Protein-A Sepharose in 30 mM Tris, 20 150 mM NaCI, and 0J % (volume /volume) Tween 20 (pH 7.4). The bottoms of the filtration units were punched out and counted in a scintillation counter (Packard Tri-carb 4530). To test the autoantigenic properties of the purified GADg 5 , increasing concentrations of unlabelled protein was added to sera incubated with 35 S- 25 methionine labelled GAD 65 (see Table 6, below). The immuneprecipitates were either counted directly or submitted to SDS-page and autoradiography. Values are mean of 3 experiments ± s.d. Serum + 10 μl buffer: 3123 cpm. Table 6: Displacement of 35 S-Met-GADg 5 with purified GADg 5 . ng GAD 65 cpm ± s.d. 670 648 ± 10.6 67 669 ± 43.7 13 777 ± 73.0 2.9 865 ± 52.6 0.54 1222 ± 18.3 0.11 1985 ± 54.2 0.022 2718 ± 243 0.0043 2815 ± 87.3 To characterize the precipitated antigen (see Figure 3), the Protein-A Sepharose with bound immunecomplexes was washed 5 times by centrifugation in 50 mM Tris, 150 mM NaCI, and 0J % (volume/volume) Tween 20 (pH 7.4), and boiled for 3 minutes in 1 M Tris, 10 mM DTT, 60 % (weight/volume) sucrose, 15 % (weight/volume) SDS, and 0.01 % (weight/volume) bromophenol blue (pH 6.8). The supernatant after centrifugation was submitted to SDS-page (7.5 %) and autoradiography (Medical X-ray film, Fuji, Japan). 60 μl were applied each lane. 10 IDDM and 10 control sera (2μl, 1 :25) were immunoprecipitated with in vitro translated 35 S-GAD 65 and further displaced with 10 μl (670 ng) of recombinant GADg (see Table 7, below). Table 7: Immunoprecipitation with in vitro translated GADg 5 . Serum cpm with GAD 65 cpm without GAD^ IDDM 1 785 3990 IDDM 2 595 2691 IDDM 3 723 3566 IDDM 4 658 3494 IDDM 5 692 3692 IDDM 6 584 2843 IDDM 7 574 3835 IDDM 8 625 4193 IDDM 9 589 4036 IDDM 10 709 3061 Control 1 605 533 Control 2 941 779 Control 3 819 717 Control 4 702 594 Control 5 762 762 Control 6 957 716 Control 7 700 718 Control 8 1011 892 Control 9 942 874 Control 10 993 825 Example 9: Enzyme Trapping Assay To 100 μl of purified GADg 5 (6.7 μg/ml) were added 25 μl of protein A Sepharose purified GAD1 (2.0 mg/ml) diluted 2 to 256 fold in 50 mM potassium phosphate, 1 mM EDTA, 1 % (volume/volume) TX-114, 0.2 mM PLP, and 1 mM AET (pH 7.2). 25 μl of buffer were added to one sample as a control. Samples were gently rotated over night at 4°C. 4 mg of Protein A Sepharose washed twice in the above buffer were added to each sample and incubated under gentle rotation for two hours at room temperature. The suspensions were centrifuged at 3000 x g for 2 minutes. Three aliquots of 25 μl of each sample were taken for enzyme activity measurement. A similar experiment was carried out using the GAD6 monoclonal antibody. To test the antigenic properties of the purified recombinant material, we looked at the ability of a monoclonal antibody GAD1 (which does not recognize denatured GAD 65 ) to remove the enzymatic activity from the incubation medium by immunoprecipitation. The binding of enzyme active GAD 65 to GAD1 was concentration dependent (see Table 8, below). Table 8: Trapping of enzymatic activity with GAD 65 . μg GAD-1 mU/ml ± s.d. 28.8 0.074 ± 0.0017 14.4 0.067 ± 0.012 7J9 0.043 ± 0.004 3.59 0.037 ± 0.014 1.80 0.032 ± 0.0015 0.90 0.332 ± 0.0075 0.45 0.603 ± 0.0446 0.22 0.712 ± 0.104 control 0.679 ± 0.120 At a GAD1 dilution of 1 :32 (1.8 μg/ml), the enzymatic activity in the supernatant was 0.0 U/ml corresponding to a specific binding capacity of 5.8 μg GAD1/mU GADg5. In a similar experiment using the monoclonal antibody GAD6 (Dr. D.I. Gottlieb and the Development Studies Hybridoma Bank, N01-HD-6-2915) recognizing denatured as well as native GADg 5 , a similar figure of 7.32 μg GAD6/ mU GAD 65 was calculated.

Claims: CLAIMS 1. Glutamic acid decarboxylase 65 compositions containing a reducing agent and/or a detergent and/or an alcohol and/or a divalent metal ion. 2. Composition according to Claim 1 , characterized in that the content of GAD 65 therein is in the range from about 0.01 mg/ml to about 10 mg/ml, preferably in the range from about 0J mg/ml to about 3 mg/ml, and more preferred in the range from about 1 to about 3 mg/ml. 3. Composition according to Claim 1 or 2, characterized in that it contains GAD in very pure form, preferably GAD having a purity of more than about 95 % (weight/weight) relative to the total amount of protein. 4. Composition according to any one of the preceding claims, characterized in that it contains a reducing agent. 5. Composition according to the previous claim, characterized in that the reducing agent is dithiothreitol (DTT), 2-mercaptoethanol, cysteine or cystamine. 6. Composition according to any one of the two preceding claims, character¬ ized in that the reducing agent is present in a concentration in the range from 1 mM to about 100 mM, preferably in a concentration less than about 20 mM. 7. Composition according to any one of the Claims 1 through 3, characterized in that it contains a detergent. 8. Composition according to the previous claim, characterized in that the detergent is TX-114, TX-100, n-octyl glucoside or SDS. 9. Composition according to any one of the two previous claims, characterized in that the detergent is present in a concentration in the range from about 0.01 % to about 10 % (volume/volume or weight/ volume), preferably in a concentration in the range from about 0.5 % to about 5 % (volume/volume or weight/volume), if the detergent is TX-114, TX-100 or n-octyl glucoside; and in a concentration in the range from about 0.001 % to about 1 % (volume/volume or weight/volume), preferably in the range from about 0.01 % to about 0.1% (volume/volume or weight/volume), if the detergent is SDS. 10. Composition, according to any one of Claims 1 through 3, characterized in that it contains an alcohol. 11. Composition according to the previous claim, characterized in that the alcohol is glycerol or a polyethylene glycol, for example, PEG 600. 12. Composition according to any one of the two preceding claims, characterized in that the alcohol is present in a concentration in the range from about 20 % to about 70 % (volume/volume), preferably in a concentration less than about 50 % (volume/volume). 13. Composition according to any one of Claims 1 through 3, characterized in that it contains a divalent metal ion. 14. Composition according to the preceding claim, characterized in that the divalent metal ion is a nickel or zinc ion. 15. Composition according to any one of the two preceding claims, characterized in that the divalent metal ion is present in a concentration in the range from about 1 mM to about 10 mM. 16. A process for isolation or purification of GAD β5 , characterized in that during the isolation or purification procedure a reducing agent is present. 17. A process according to the preceding claim, characterized in that the reducing agent is one of the compounds mentioned in Claim 5. 18. Any novel feature or combination of features described herein.

Inventors: HEJNAES KIM, MARSHALL MICHAEL OWEN, MOODY ALISTER J, HEJNAES, KIM, MARSHALL, MICHAEL, OWEN, MOODY, ALISTER, J.
Applicants: HEJNAES KIM, MARSHALL MICHAEL OWEN, MOODY ALISTER J, NOVONORDISK AS, HEJNAES, KIM, MARSHALL, MICHAEL, OWEN, MOODY, ALISTER, J., NOVO NORDISK A/S
Application year: 1994
Main classification: C12N9
IPC codes: C12N9